A method for detecting a target nucleic acid having a target sequence in a sample, which has been used, includes a hybridization method in which a probe is used, a PCR method in which oligonucleotide primers are used, and other methods. Further, the PCR method is generally used in various fields including the detection and cloning of target nucleic acid, and various improved methods have been developed.
A so-called real time PCR has been known, which is a PCR method that performs amplification of a target sequence and analysis of the amplified product simultaneously. Means for analyzing the amplified products that has been known include, for example, a Taq-Man probe method (U.S. Pat. No. 5,210,015 A, JP 06-500021 A, and Holland et al., Pro. Natl. Aca. Sci. USA., 88, 7276-7280, 1991), a molecular beacon method (JP 05-123195 A, Sanjay Tyagi et al., Nature Biotechnology, vol 14, March 1996), an intercalator method (Bio/Technology, 10, 413-417, 1992, Bio/Technology, 11, 1026-1030, 1993, and JP05-237000 A), and the like.
In the Taq-Man probe method, a fluorescent material and a probe labeled with a quencher that quenches fluorescence emitted by the fluorescent material are used. When the probe is hybridized with a target nucleic acid, the quencher quenches the fluorescence while the probe is cleaved by the 5′→3′ exonuclease activity of the polymerase used in PCR at the time of amplification reaction. As a result, the fluorescent material is released from the quencher to emit fluorescence. The amount of the double stranded DNA molecule can be known from this fluorescence.
Further, the molecular beacon method is a method that uses a probe including a sequence complementary to a target sequence and an arm having sequences complementary to each other at both sides thereof as well as a fluorescent material and a quencher bonded to both the ends. When the probe is annealed to the target nucleic acid, the fluorescent material emits fluorescence while when the probe is dissociated from the target nucleic acid, the probe forms an arm resulting in that the fluorescent material and the quencher become closer to each other to cause quenching.
On the other hand, the intercalator method is a method that detects a double stranded DNA using an intercalator such as ethidium bromide.
Although the methods for quantifying PCR amplified products in real time have been known as described above, they have problems; the Taq-Man probe method cannot be applied in the case of amplification methods that use polymerases having no 5′→3′ exonuclease activity, the molecular beacon method is difficult to design a probe and suffers poor detection efficiency due to the intermolecular bond, and the intercalator method has no sequence specificity.